PROJECT SUMMARY von Willebrand factor (VWF) is a key component of the blood coagulation system and the plasma chaperone for factor VIII (FVIII). Deficiencies of VWF result in the most common inherited bleeding disorder in humans, von Willebrand disease (VWD) whereas loss of VWF proteolytic processing by ADAMTS13 results in thrombotic thrombocytopenic purpura (TTP). In addition, elevated levels of plasma VWF are also a major risk factor for thrombosis. This competing renewal application will continue our laboratory?s longstanding focus on the genetic basis for VWD and TTP, exploiting recent transformative advances in genetic and genomic technology to uncover novel pathways contributing to the control of VWF and ADAMTS13 function and laying the foundation for a ?precision medicine? approach to the diagnosis and management of these disorders. A novel VWF regulatory gene mapped to a segment of human chromosome 2 through previous human genetic studies will be identified through detailed genomic sequence analysis and plasma VWF measurements in an additional large cohort of human subjects. The function of this gene will be explored through expression analysis of the sequence variants identified in human subjects, and modeling by CRISPR/Cas9 ?genome editing? in laboratory mice. Similar tools will be used to characterize a novel modifier gene for TTP susceptibility in the mouse previous localized to mouse chromosome 5. The specific genomic variant(s) responsible for this difference in TTP susceptibility will be identified by the generation of mice carrying a series of gene targeted alterations across this chromosome 5 candidate region, and the function of the responsible gene characterized. Finally, this project will assemble a comprehensive dataset for the functional impact of all possible single amino acid substitutions within the VWF A1 and A2 domains to provide a complete inventory of potential human mutations causing type 2A and 2B VWD, the 2 most common qualitative VWD variants. These data will address the increasingly important clinical problem of ?variant of uncertain significance?, a key challenge for the entire field of human genetics. We anticipate that these data will be a valuable resource for properly categorizing and interpreting VWF gene variants. These data should begin to lay the foundation for eventual diagnosis and subclassification of VWD type 2 on the basis of DNA sequence alone, with the results also accurately guiding therapy and enabling true ?precision medicine?. This work could also serve as a useful paradigm for other genetic diseases and future high throughput genetic diagnoses, as we enter the era of widespread clinical whole genome/exome sequencing. Taken together, this research program will apply cutting-edge genetic and genomic technologies to identify critical genes modifying plasma VWF levels in humans, as well as yielding information about fundamental VWF and ADAMTS13 biology that could lay the ground work for future novel approaches to the diagnosis and treatment of VWD, TTP, and other hemostatic disorders.